Effect of empagliflozin on ectopic fat stores and myocardial energetics in type 2 diabetes: the EMPACEF study.

BACKGROUND: Empagliflozin is a sodium-glucose cotransporter 2 (SGLT2) inhibitor that has demonstrated cardiovascular and renal protection in patients with type 2 diabetes (T2D). We hypothesized that empaglifozin (EMPA) could modulate ectopic fat stores and myocardial energetics in high-fat-high-sucrose (HFHS) diet mice and in type 2 diabetics (T2D). METHODS: C57BL/6 HFHS mice (n = 24) and T2D subjects (n = 56) were randomly assigned to 12 weeks of treatment with EMPA (30 mg/kg in mice, 10 mg/day in humans) or with placebo. A 4.7 T or 3 T MRI with 1H-MRS evaluation-myocardial fat (primary endpoint) and liver fat content (LFC)-were performed at baseline and at 12 weeks. In humans, standard cardiac MRI was coupled with myocardial energetics (PCr/ATP) measured with 31P-MRS. Subcutaneous (SAT) abdominal, visceral (VAT), epicardial and pancreatic fat were also evaluated. The primary efficacy endpoint was the change in epicardial fat volume between EMPA and placebo from baseline to 12 weeks. Secondary endpoints were the differences in PCr/ATP ratio, myocardial, liver and pancreatic fat content, SAT and VAT between groups at 12 weeks. RESULTS: In mice fed HFHS, EMPA significantly improved glucose tolerance and increased blood ketone bodies (KB) and ß-hydroxybutyrate levels (p < 0.05) compared to placebo. Mice fed HFHS had increased myocardial and liver fat content compared to standard diet mice. EMPA significantly attenuated liver fat content by 55%, (p < 0.001) but had no effect on myocardial fat. In the human study, all the 56 patients had normal LV function with mean LVEF = 63.4 ± 7.9%. Compared to placebo, T2D patients treated with EMPA significantly lost weight (- 2.6 kg [- 1.2; - 3.7]) and improved their HbA1c by 0.88 ± 0.74%. Hematocrit and EPO levels were significantly increased in the EMPA group compared to placebo (p < 0.0001, p = 0.041). EMPA significantly increased glycosuria and plasma KB levels compared to placebo (p < 0.0001, p = 0.012, respectively), and significantly reduced liver fat content (- 27 ± 23 vs. - 2 ± 24%, p = 0.0005) and visceral fat (- 7.8% [- 15.3; - 5.6] vs. - 0.1% [- 1.1;6.5], p = 0.043), but had no effect on myocardial or epicardial fat. At 12 weeks, no significant change was observed in the myocardial PCr/ATP (p = 0.57 between groups). CONCLUSIONS: EMPA effectively reduced liver fat in mice and humans without changing epicardial, myocardial fat or myocardial energetics, rebutting the thrifty substrate hypothesis for cardiovascular protection of SGLT2 inhibitors. Trial registration NCT, NCT03118336. Registered 18 April 2017, https://clinicaltrials.gov/ct2/show/NCT03118336.

Pheromone dispensers, including organic polymer fibers, described in the crop protection literature: comparison of their innovation potential.

Pheromone dispensers, although known in a variety of different designs, are one of the few remaining technical bottlenecks along the way to a sustainable pheromone based strategy in integrated pest management (IPM). Mating disruption with synthetic pheromones is a viable pest management approach. Suitable pheromone dispensers for these mating disruption schemes, however, are lagging behind the general availability of pheromones. Specifically, there is a need for matching the properties of the synthetic pheromones, the release rates suitable for certain insect species, and the environmental requirements of specific crop management. The "ideal" dispenser should release pheromones at a constant but pre-adjustable rate, should be mechanically applicable, completely biodegradable and thus save the costs for recovering spent dispensers. These should be made from renewable, cheap organic material, be economically inexpensive, and be toxicologically and eco-toxicologically inert to provide satisfactory solutions for the needs of practicing growers. In favourable cases, they will be economically competitive with conventional pesticide treatments and by far superior in terms of environmental and eco-toxicological suitability. In the course of the last 40 years, mating disruption, a non-toxicological approach, provided proof for its potential in dozens of pest insects of various orders and families. Applications for IPM in many countries of the industrialized and developing world have been reported. While some dispensers have reached wide circulation, only few of the key performing parameters fit the above requirements ideally and must be approximated with some sacrifice in performance. A fair comparison of the innovation potential of currently available pheromone dispensers is attempted. The authors advance here the use of innovative electrospun organic fibers with dimensions in the "meso" (high nano- to low micrometer) region. Due to their unique multitude of adjustable parameters, they hold considerable promise for future pest control against a variety of pest insects. In combination with well known synthetic sex pheromones, they can be used for communication disruption studies. One example, the pheromone of the European grape vine moth Lobesia botrana (Lepidoptera: Tortricidae), in combination with Ecoflex fibers, has been thoroughly tested in vineyards of Freiburg, Southwest Germany, with promising results. Seven weeks of communication disruption have been achieved, long enough to cover any one of several flights of this multivoltine grape pest. Disruption effects of around 95% have been achieved which are statistically indistinguishable from positive controls tested simultaneously with Isonet LE fibers, while an untreated negative control is significantly different. Ecoflex is a cheap organic co-polyester and completely biodegradable within half a year. Thus, an extra recovery step as with some other dispensers is unnecessary. This co-polyester is also of proven non-toxicity. The extension of the seven week disruption period towards half a year (the entire duration of all 3 Lobesia flights combined) is desirable and is under additional investigation in the near future. The discovery of suitable mesofibers is protected by European and US patents. The pheromone literature appearing between 1959 and today contains more than 25,000 references. This wealth of information is immediately applicable to pest management. It has major impacts on chemical ecology and IPM. In this paper, an attempt is made to compare the systems described in the literature and to derive some predictions about their prospective innovation potential. Special emphasis is given to the new development of organic biodegradable microfibers. To this end, a new electronic searching algorithm is introduced for reviewing the entries to be found in 4 specific databases. Its prominent features will be described. Surprisingly we found no previous entries in the literature linking pheromones with biodegradable organic polymer fibers whose diameters are in the dimension range of low micrometers and in the upper nanometer scale. In conclusion, the microfiber-pheromone combination must be considered as a novel approach whose virtues should be further explored for IPM in the near future.

New dispenser types for integrated pest management of agriculturally significant insect pests: an algorithm with specialized searching capacity in electronic data bases.

Pheromone effects discovered some 130 years, but scientifically defined just half a century ago, are a great bonus for basic and applied biology. Specifically, pest management efforts have been advanced in many insect orders, either for purposes or monitoring, mass trapping, or for mating disruption. Finding and applying a new search algorithm, nearly 20,000 entries in the pheromone literature have been counted, a number much higher than originally anticipated. This compilation contains identified and thus synthesizable structures for all major orders of insects. Among them are hundreds of agriculturally significant insect pests whose aggregated damages and costly control measures range in the multibillions of dollars annually. Unfortunately, and despite a lot of effort within the international entomological scene, the number of efficient and cheap engineering solutions for dispensing pheromones under variable field conditions is uncomfortably lagging behind. Some innovative approaches are cited from the relevant literature in an attempt to rectify this situation. Recently, specifically designed electrospun organic nanofibers offer a lot of promise. With their use, the mating communication of vineyard insects like Lobesia botrana (Lep.: Tortricidae) can be disrupted for periods of seven weeks.

Antibodies to normal human melanocytes in vitiligo.

Most patients with active vitiligo (82% of 61) have antibodies to antigens of normal human melanocytes that can be detected by specific immunoprecipitation of radioiodinated, detergent-soluble, melanocyte macromolecules. Such antibodies were present in only 12% of patients with melanoma and in none of 35 patients with nonpigmentary skin diseases. The antibodies were directed to a common antigen(s) on melanocytes that was not present on normal fibroblasts or keratinocytes. These observations suggest that vitiligo is an autoimmune disease mediated by antibodies to melanocyte-associated antigen(s).

Cell surface antigens of human melanocytes and melanoma. Expression of adenosine deaminase binding protein is extinguished with melanocyte transformation.

It has been proposed that the pathogenesis of melanoma proceeds through multiple stages, ranging from benign proliferation of melanocytic cells to acquisition of the capacity to invade tissues and metastasize. During investigations of cell surface antigens expressed by melanocytes and melanoma, we identified an antigen system that was expressed by cultured normal melanocytes but not by melanoma cell lines. mAbs against this antigen detected a 120-kD cell surface glycoprotein on melanocytes. This molecule had been identified previously as the binding protein for adenosine deaminase (ADAbp). ADAbp was expressed by 51 melanocyte cell lines derived from normal fetal, newborn, and adult skin and adult choroid, but not by 102 melanoma cell lines derived from primary and metastatic lesions. Studies with radiolabeled bovine adenosine deaminase, confirmed that melanocytes expressed binding sites for adenosine deaminase, but no binding sites were detected on cultured melanoma cells. Further studies showed that ADAbp+ melanocytes became ADAbp- upon malignant transformation in vitro. Immunohistochemical studies on a panel of frozen tissues demonstrated reactivity of anti-ADAbp mAbs with epidermal melanocytes and benign junctional nevi, but not with potentially premalignant dysplastic nevi or primary/metastatic melanoma lesions. These studies demonstrate that ADAbp expression is lost with malignant transformation of melanocytes, presumably at an early stage in the transformation process.

Surface antigens of melanocytes and melanomas. Markers of melanocyte differentiation and melanoma subsets.

The surface antigens of melanocytes from newborn and adult skin have been analyzed with monoclonal antibodies detecting cell surface antigens of malignant melanoma. Antigenic markers that distinguish early, intermediate, and mature stages in melanocyte differentiation have been defined. The characteristics of the normal melanocyte precursor have been inferred from the features of melanomas that express early markers of melanocyte differentiation. A rudimentary surface antigen map of cells undergoing melanocyte differentiation and a new classification of melanomas based on the expression of melanocyte differentiation antigens are proposed.

Class II histocompatibility antigen expression in human melanocytes transformed by Harvey murine sarcoma virus (Ha-MSV) and Kirsten MSV retroviruses.

Human melanocytes infected with Ki-MSV or Ha-MSV, but not amphotropic MuLV, undergo a series of transformation-related changes that are characteristic of malignant melanoma. These are (a) expression of Ia antigens, in particular DP, DQ, and DR class II histocompatibility gene products, (b) a transformed morphology and ability to grow in soft agar, and (c) a 5-10-fold increase in the cell surface expression of GD3 ganglioside. However, other characteristics of melanoma, such as independence from specific growth factors and loss of adenosine deaminase binding protein were not observed. We conclude that viral ras oncogenes initiate early transformation events in melanocytes, and that Ia antigen expression is a transformation marker in this system.

Holocaust survivors: the pain behind the agony. Increased prevalence of fibromyalgia among Holocaust survivors.

OBJECTIVES: To assess the frequency of fibromyalgia among a population of Holocaust survivors in Israel as well as the occurrence of post-traumatic stress disorder (PTSD) and concurrent psychiatric symptoms, including depression and anxiety among survivors. METHODS: Eighty-three survivors of the Nazi Holocaust and 65 age-matched individuals not exposed to Nazi occupation were recruited. Physical examination and manual tender point assessment was performed for the establishment of the diagnosis of fibromyalgia and information was collected regarding quality of life (SF-36), physical function and health (FIQ), psychiatric symptoms (SCL-90) and PTSD symptoms (CAPS). RESULTS: Significantly increased rates of fibromyalgia were identified among Holocaust survivors compared with controls (23.81% vs. 10.94, p<0.05). Significantly increased rates of posttraumatic symptoms and measures of mental distress were also identified among survivors. CONCLUSIONS: The results indicate a significantly increased prevalence of fibromyalgia among Holocaust survivors six decades after the end of the Second World War. This finding furthers our knowledge regarding the long-term effect of stress on the development of fibromyalgia.

Growth regulation of human melanocytes: mitogenic factors in extracts of melanoma, astrocytoma, and fibroblast cell lines.

Melanocytes derived from fetal or adult skin do not propagate in vitro unless cultured in the presence of factors such as 12-O-tetradecanoylphorbol 13-acetate (TPA). In a search for physiological factors regulating the growth of melanocytes, extracts of various cultured cell types were tested. Factors produced by melanoma and astrocytoma cell lines support continued proliferation of melanocytes in the absence of TPA. WI-38, a fibroblast cell line derived from human embryonic lung, was the most active source of melanocyte growth factors. No melanocyte growth-promoting activity was found in extracts of cultured neuroblastoma, renal cancer, normal keratinocytes, or renal epithelium. Nerve growth factor, epidermal growth factor, melanocyte-stimulating hormone, transforming growth factor-beta, and platelet-derived growth factor did not have growth-promoting activity for melanocytes. The presence of melanocyte growth factors and TPA together resulted in the strongest mitogenic activity for melanocytes, permitting the recovery (at 20 days) of 4 to 20 times as many cells as in growth factor or TPA alone.

Early endothelial dysfunction in young type 1 diabetics.

OBJECTIVES: Endothelial dysfunction is a known precursor of atherosclerosis and can be assessed by measuring the brachial artery flow-mediated dilatation (FMD) via ultrasonography. This study investigated endothelial function in young type 1 diabetics without cardiovascular morbidity or diabetes-related pathology. METHODS: Young diabetics and healthy controls were recruited, both meeting strict inclusion and exclusion criteria. To prove absence of subclinical atherosclerosis, intima-media thickness (IMT) measurements at the carotid bifurcation were done in all of them. FMD was measured at the brachial artery. The results were compared using the t-test and the influences of different variables on FMD were assessed using multiple linear regression. RESULTS: Twenty-six diabetics (23.4+/-5.8 years) and 36 healthy volunteers (23.1+/-2.8 years) were recruited. The duration of diabetes was 9.2+/-5.3 years; metabolic control was moderate (HbA1c 7.6+/-1.0%) and IMT was normal in both groups. FMD was significantly impaired in type 1 diabetics (7.13+/-0.43 vs. 8.77+/-0.43%; p=0.002). The FMD grade was associated with diabetes and age. Patients with a good metabolic control (HbA1c

Active site rearrangement and structural divergence in prokaryotic respiratory oxidases.

Cytochrome bd-type quinol oxidases catalyze the reduction of molecular oxygen to water in the respiratory chain of many human-pathogenic bacteria. They are structurally unrelated to mitochondrial cytochrome c oxidases and are therefore a prime target for the development of antimicrobial drugs. We determined the structure of the Escherichia coli cytochrome bd-I oxidase by single-particle cryo-electron microscopy to a resolution of 2.7 angstroms. Our structure contains a previously unknown accessory subunit CydH, the L-subfamily-specific Q-loop domain, a structural ubiquinone-8 cofactor, an active-site density interpreted as dioxygen, distinct water-filled proton channels, and an oxygen-conducting pathway. Comparison with another cytochrome bd oxidase reveals structural divergence in the family, including rearrangement of high-spin hemes and conformational adaption of a transmembrane helix to generate a distinct oxygen-binding site.

Identification of an immunologically distinct papillomavirus from lesions of epidermodysplasia verruciformis.

Virions isolated from warts of 2 siblings with epidermodysplasia verruciformis (EV), a rare disease characterized by the lifelong growth of warty skin tumors containing papova-like virions, were compared to isolates of human papillomavirus (HPV) from 3 pools of plantar and common hand warts. The length of relaxed, circular (form II) molecules of EV virion DNA approximated the length of HPV DNA but was 3.3% longer. Antisera prepared in rabbits against the 3 HPV pools coated and aggregated HPV in immune electron microscopy (IEM) tests but did not react with EV virions. These antisera reacted at high titers in complement fixation (CF) tests with HPV and reacted only weakly in CF tests with EV virions. Rabbit antisera to EV virions coated and aggregated EV virions but reacted only weakly or not at all with HPV virions in IEM tests. These sera reacted in CF with EV virions only. The data indicated that virions from the EV patients represent an immunologically distinct papillomavirus.

Binding of phorbol dibutyrate and epidermal growth factor to cultured human epidermal cells.

Primary cell cultures of normal human epidermal keratinocytes and melanocytes and human cell lines established from a primary melanoma (SK-PM-4) and metastatic melanomas (HO#1, SK-MEL21, and SK-MEL37) contain specific and saturable receptors for the tumor promoter phorbol dibutyrate (PDBu). Scatchard analyses of the keratinocytes revealed two classes of binding sites: 1) a high-affinity class (affinity constant = 37 nM; 1.3 X 10(6) sites/cell) and a low-affinity class (affinity constant = 4,880 nM; 7 X 10(7) sites/cell). The melanoma cultures, likewise, showed high- and low-affinity classes of PDBu binding sites. However, the affinity constant values and total numbers of sites in the melanoma cells were lower than the corresponding values in the keratinocytes. The binding of [3H]PDBu to human keratinocytes was inhibited by the tumor promoters 12-O-tetradecanoylphorbol 13-acetate and teleocidin but not by phorbol, which lacks tumor-promoting activity. Human serum also inhibited binding. Specific receptors for epidermal growth factor (EGF) were demonstrated in the keratinocytes and primary melanoma cultures. In contrast, three metastatic melanoma cultures gave negligible levels of EGF binding. Among the various cell types, the extent of [3H]PDBu binding did not correlate with the extent of EGF binding, indicating that these two substances occupy distinctly separate types of receptors.

Glycoproteins as differentiation markers in human malignant melanoma and melanocytes.

Human malignant melanoma cell lines have been divided into three broad groups on the basis of morphology, pigmentation, tyrosinase levels, the 2-dimensional electrophoretic patterns of their [3H]glucosamine-labeled glycoproteins and the presence or absence of an extracellular matrix of fibronectin. The most pigmented cell lines were characterized by the synthesis of a novel glycoprotein with a molecular weight of 75,000 and the absence of a fibronectin matrix. As cultured skin melanocytes also had these characteristics, this group of melanomas appears to be the most differentiated. Melanoma cell lines in the amelanotic group were characterized by the synthesis of high levels of HLA-DR antigen and by the production of an extracellular fibronectin matrix.

Detection of antibodies to melanocytes in vitiligo by specific immunoprecipitation.

Immunoprecipitation was used to assay for antibodies to normal human melanocytes in the sera of 12 patients with common vitiligo and 12 normal individuals. The procedure is based on the specific immunoprecipitation using protein A-sepharose of antibodies binding to detergent-soluble, radioiodinated macromolecules of normal human melanocytes grown in culture. Antibodies to melanocytes were found in all 12 patients with vitiligo but in none of the normal sera. None of the sera reacted specifically to normal human fibroblasts or to human melanoma cells radioiodinated in a similar manner. These observations suggest that antibodies to melanocyte-associated antigens are present in common vitiligo.

Decreased expression of epidermal cytoplasmic antigens in cultured human keratinocytes.

The expression of upper cytoplasmic (U-CYT) antigens which are expressed only in the superficial layers of the epidermis and are markers of epidermal cell differentiation in vivo and of basement zone (BMZ) antigens reacting with bullous pemphigoid serum was studied in keratinocytes in tissue culture. The cells were cultured at an acid pH (5.6-5.8) similar to that of skin and without feeder cells, dermal tissue, or collagen. It was found that the expression of U-CYT antigens decreased markedly in culture. These antigens were expressed in 45-65% of epidermal cells prepared from fresh skin, but in only 5-10% of cells which had been grown in primary culture over 1 mo, and in no cells in secondary or tertiary culture. By contrast, BMZ antigens continued to be expressed in culture. These antigens were expressed by 20-35% of epidermal cells prepared from fresh tissue and by 15-35% of keratinocytes in primary, secondary or tertiary culture. These findings indicate that U-CYT and BMZ antigens can be used to type subpopulations of human keratinocytes in suspension, and suggest that the differentiation of these cells in vitro differs from that which occurs in vivo.

Differentiation and shedding of surface macromolecules of human keratinocytes.

Macromolecules expressed on the external surface of human keratinocytes in tissue culture were radioiodinated by the lactoperoxidase technique and solubilized by lysing the cells in nonionic detergent. Soluble labeled proteins were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pattern of proteins expressed by keratinocytes was distinct for these cells. The major labeled components had Mrs of 28K, 40K, 68K, 72K, and 77K. The expression of several macromolecules changed with the length of time the cells were in culture, suggesting that their expression was linked to epidermal differentiation. Lastly, many but not all of the macromolecules expressed on the surface of keratinocytes were selectively and rapidly released into medium by the cells.

An alternative approach to depigmentation by soybean extracts via inhibition of the PAR-2 pathway.

The protease-activated receptor 2, expressed on keratinocytes but not on melanocytes, has been ascribed functional importance in the regulation of pigmentation by phagocytosis of melanosomes. Inhibition of protease-activated receptor 2 activation by synthetic serine protease inhibitors requires keratinocyte-melanocyte contact and results in depigmentation of the dark skinned Yucatan swine, suggesting a new class of depigmenting mechanism and agents. We therefore examined natural agents that could exert their effect via the protease-activated receptor 2 pathway. Here we show that soymilk and the soybean-derived serine protease inhibitors soybean trypsin inhibitor and Bowman-Birk inhibitor inhibit protease-activated receptor 2 cleavage, affect cytoskeletal and cell surface organization, and reduce keratinocyte phagocytosis. The depigmenting activity of these agents and their capability to prevent ultraviolet-induced pigmentation are demonstrated both in vitro and in vivo. These results imply that inhibition of the protease-activated receptor 2 pathway by soymilk may be used as a natural alternative to skin lightening.

Inhibition of melanosome transfer results in skin lightening.

The chemical basis of melanogenesis is well documented, but the mechanism of melanosome transfer and the regulation of pigmentation by keratinocyte-melanocyte interactions are not well understood. Therefore we examined the effects of serine protease inhibitors on skin pigmentation and found that the protease-activated receptor 2, expressed on keratinocytes, may regulate pigmentation via keratinocyte-melanocyte interactions. Here we show that modulation of protease-activated receptor 2 activation affects melanosome transfer into keratinocytes, resulting in changes in pigment production and deposition. SLIGRL, the protease-activated receptor 2 activating peptide, enhanced melanosome ingestion by keratinocytes, thus increasing pigment deposition. RWJ-50353, a serine protease inhibitor, led to reduced pigment deposition in melanocytes and depigmentation. Electron microscopy studies illustrated an accumulation of immature melanosomes inside melanocytes and abnormal dendrite dynamics in RWJ-50353-treated epidermal equivalents. RWJ-50353 induced a visible and dose-dependent skin lightening effect in the dark-skinned Yucatan swine. Examinations by electron microscopy indicated that the in vivo transfer of melanosomes from melanocytes to keratinocytes was affected. Our data suggest that modulation of keratinocyte-melanocyte interactions via the protease-activated receptor 2 pathway affects melanosome transfer. The use of RWJ-50353 to modulate protease-activated receptor 2 activation could lead to a new class of depigmenting agents.

Immunoelectron microscopic identification of Langerhans cells using a new antigenic marker.

The specificity of a monoclonal antibody (OKT6) for epidermal Langerhans cells was examined by immunoelectron microscopy. Peroxidase-labeled OKT6 bound to 1-5% of suspended human epidermal cells, as determined by light microscopy. Electron microscopic examination of peroxidase-labeled cells revealed that all Birbeck granule-containing Langerhans cells bound OKT6. In addition, a small population of indeterminate cells, lacking the Birbeck granule, was also labeled with OKT6. The ultrastructural studies confirm the specificity of OKT6 for Langerhans cells and suggest that the indeterminate cell represents a related cell population.